(a) Field of the Invention
The invention relates to cyclic compounds having a 3-dimensional structure which binds to neurotrophin receptors, their uses for the treatment or prevention of peripheral and central nervous system diseases, neuromas at the end of an amputated limb and neoplastic diseases which express neurotrophin receptors.
(b) Description of Prior Art
Nerve Growth Factor (NGF) is a protein which has prominent effects on developing sensory and sympathetic neurons of the peripheral nervous system and cholinergic neurons of the CNS. NGF is a polypeptide growth factor member of the neurotrophin family, which includes Brain Derived Neurotrophic Factor (BDNF), Neurotrophin-3 (NT3) and Neurotrophin-4/5 (NT-4/5). NGF controls the survival and development of certain neuronal populations and has been reported to be a mitogen for other cell types. Two cell surface NGF receptors have been characterized on the basis of binding affinity and signal transduction properties, namely the p75 low affinity NGF receptor (LNGFR) and p140 trkA.
The p75 receptor (Kd=10.sup.-9 M; Johnson, D. et al. (1986) Cell, 47: 545-554) is a 75 kDa glycoprotein member of the TNFR/Fas/CD40 family of receptors (Itoh et al., (1991) Cell, 66:233-243). p75 contains no intrinsic catalytic activity but can associate with the ERK family of soluble kinases (Volonte C. et al. (1993) J. Biol. Chem., 268:21410-21415), and plays a role in protection from neuronal apoptotic death (Rabizadeh, S. et al. (1993) Science, 261:345-348). p75 is also the low affinity binding receptor for BDNF and NT-3 (Ibanez, C. F. et al. (1991) Eur. Mol. Biol. Org. J., 10:2105-2110) but these latter growth factors each have distinct trk receptors.
The p140 trkA receptor is a 140 kDa glycoprotein encoded by the trk proto-oncogene (Klein, R. et al. (1991) Cell, 65:189-197). Scatchard analysis of cells expressing only trkA receptors showed a curvilinear plot with Kd .sup..about. 10.sup.-11 M and 10.sup.-9 M (Jing, S. et al. (1992) Neuron, 9: 1067-1079). The trkA receptor has intrinsic tyrosine kinase activity and is capable of evoking cellular neurotrophic responses in vitro in the absence of p75 LNGFR (Hempstead, B. L. et al. (1991) Nature, 350: 678-683).
The co-expression of both p75 and p140 allows detection of a higher affinity NGF receptor and affords a Kd .sup..about. 10.sup.-12 M (Jing, S. et al. (1992) Neuron, 9: 1067-1079). Hence, p75 can associate with different trk-receptors to form high affinity binding sites but neurotrophin binding specificity is mediated by distinct trk-receptors (Ip, N.Y. et al. (1993) Neuron, 10:137-149). The molecular nature of the functional receptor remains unknown.
The NGF protein has been purified, cloned and sequenced (Angeletti, R. H. et al. (1973) Biochemistry, 12: 100-115). Cloning of NGF from different species has shown high amino acid sequence homology, and cross-species biological reactivity.
The structure of mouse NGF has been resolved from crystallographic data at 2.3 .ANG. resolution (McDonald, N. Q. et al. (1991) Nature, 345: 411-414). In the crystals, the NGF molecule is a tightly associated dimer made up of parallel protomers of 118 amino acids. Each protomer has seven .beta.-strands forming three antiparallel pairs. The .beta.-strands are linked by four exposed regions: three .beta.-turns (termed A'-A", A'"-B, and C-D) and one series of three contiguous reverse-turns (termed B-C).
The .beta.-turn and reverse-turn regions had been noted for their hydrophilic nature and unlike the mostly conserved buried residues of the .beta.-strands the .beta.-turns have little conservation between different neurotrophins (Hallbook, F. et al. (1991) Neuron, 6: 845-858). The variability and hydrophilicity of these .beta.-turn regions has prompted the hypothesis that they may be involved in determining neurotrophin receptor specificity because several dimeric molecules use .beta.-turns for critical binding surface(s). Similarly, antibodies and other members of the immunoglobulin gene superfamily (Chothia, C. et al. (1987) J. Mol. Biol., 196:901-917) and other globular proteins (Sibanda, B. L. et al. (1989) J. Mol. Biol., 206: 759-777) use .beta.-turns to interact with complementary sequences with high affinity and specificity.
Experimental evidence using mutagenesis and chimeric molecules has sustained this early hypothesis concerning .beta.-turns of NGF. For example, a chimeric BDNF molecule expressing two .beta.-turns regions of NGF was able to induce neurite outgrowth in NGF responsive sympathetic neurons to the same extent as wild type NGF (Ibanez, C. F. et al. (1991) Eur. Mol. Biol. Org. J., 10:2105-2110).
In spite of the structural information obtained, attempts to create analogs which mimic the activity of NGF .beta.-turns have not been equally successful. Longo et al (Longo, F. et al. (1990) Cell Reg., 1:189-195), found a peptide with sequences from NGF residues 23-35 which inhibited NGF activity. However, this linear peptide did not adopt the .beta.-turn structure from which it was derived in NGF and did not affect the binding of radiolabeled NGF to receptor expressing cells. The peptides were sequence analogs rather than structural analogs of NGF. Furthermore, the concentration of peptide at which inhibition of NGF biological activity occurred (2 mM) was well within concentrations at which non-specific effects can also occur.
Murphy et al (Murphy, R. A. et al. (1993) J. Neuroscience, 13(7): 2853-2862) have used a similar approach to study linear peptides derived from NGF comprising amino acids 23-35, 59-67, 69-79, and 91-100. Some limited biological effects were seen for peptides (59-67) and (91-100) in the in vitro assays of neurite survival when suboptimal concentrations of NGF and a 1,000 fold excess of peptide with respect to NGF were used. However, these peptides did not appear to bind the receptors with high affinity and did not compete with radioactive NGF for binding. In addition, antisera raised against the linear peptides did not effectively cross-react with native NGF, further suggesting that the peptides are simply sequence analogs but not true structural analogs of NGF.
The above-mentioned prior studies have contributed to defining important regions of the NGF molecule. However, the exact combination of amino acids of NGF and the 3-dimensional structure(s) that participate in binding to p75 or trkA receptors and cause biological effects remains to be determined.
Frank M. Longo et al. (European Patent Application published under No. EP-A-335,637 on Oct. 4, 1989) have disclosed what are purported to be agonist and antagonist nerve growth factors peptides. These NGF blocking peptides, which are in fact sequence analogs, can be used to inhibit the expression of mRNA and their encoded proteins whose expression is stimulated by NGF. Again, there is no teachings or suggestions of 3-dimensional structures of native NGF or of NGF blocking peptides that participate in binding to p75 or trkA receptors. The linear peptide sequences described by Longo et al. represent synthesized fragments of NGF without teachings or concern with structural motifs, architecture, folding, or bioavailability. The inhibitory activity of these non-structural, linear peptide sequence analogs of NGF was observed at very high, non-pharmacological doses of analog (2 mM), tested versus suboptimal doses of NGF. Further, the inhibitory activity reported did not include an ability to affect either NGF binding, specific receptor binding by NGF, receptor dimerization, neurite extension by NGF responsive cells, or other physiological events associated with NGF function or receptor physiology.
It would be highly desirable to be provided with a cyclic compound having a 3-dimensional structure which binds to neurotrophin receptors, which would mimic the neurotrophin 3-D conformation.